Loss of function effect of RET mutations causing Hirschsprung disease

Abstract

We have introduced three Hirschsprung (HSCR) mutations localized in the tyrosine kinase domain of RET into the RET/PTC2 chimaeric oncogene which is capable of transforming NIH3T3 mouse fibroblasts and of differentiating pC12 rat pheochromocytoma cells. The three HSCR mutations abolished the biological activity of RET/PTC2 in both cell types and significantly decreased its tyrosine phosphorylation. By contrast, a rare polymorphism in exon 18 does not alter the transforming capability of RET/PTC2 or its tyrosine phosphorylation. These data suggest a loss of function effect of HSCR mutations which might act through a dominant negative mechanism. Our model system is therefore capable of discriminating between causative HSCR mutations and rare polymorphisms in the tyrosine kinase domain of RET.

Signalling of the Ret receptor tyrosine kinase through the c-Jun NH2-terminal protein kinases (JNKs): evidence for a divergence of the ERKs and JNKs pathways induced by Ret

“…JNK1 stimulation by RET was as e cient as that obtained by heat shock (lane +) or by cycloheximide (not shown), which are known inducers of JNK1 Coso et al, 1995a). Mutations associated with Hirschsprung's disease (HSCR) exert a`loss of function' e ect on Ret; speci®cally, a HSCR R972G substitution, in the TK domain of Ret, greatly impairs both RET/MEN2A kinase and transforming activities (Pasini et al, 1995;Carlomagno et al, 1996). As shown in Figure 5, JNK1 stimulation in COS-1 cells was signi®cantly impaired when the HSCR R972G mutation was introduced in the RET/MEN2A construct.…”

“…Consistently, RETde®cient mice show lack of enteric neurons and renal dysgenesis (Schuchardt et al, 1994), and`loss of function' RET mutations are associated with Hirschsprung's disease Edery et al, 1994;Pasini et al, 1995;Carlomagno et al, 1996), which is characterized by the congenital defect of intestinal innervation. Recently, GDNF (glial cell line derived neurotrophic factor) has been identi®ed as one functional ligand for Ret.…”

“…Activated RET isoforms are highly transforming for NIH3T3 cells (Grieco et al, 1990;Santoro et al, 1994;Asai et al, 1995) and are able to induce the expression of di erentiation markers in PC12 pheochromocytoma cells (Califano et al, 1995(Califano et al, , 1996Pasini et al, 1995). Moreover, the RET/PTC1 oncogene is able to transform rat thyroid epithelial PC Cl 3 cells, causing thyrotropin-independent growth and a loss of the di erentiated phenotype (Santoro et al, 1993), and is able to induce thyroid carcinomas in transgenic mice (Jhiang et al, 1996;Santoro et al, 1996).…”

A GTG to ATG novel point mutation at codon 804 in exon 14 of the RET proto-oncogene in two families affected by familial medullary thyroid carcinoma

“…A mutation, at the same codon 804, that causes the replacement of a Val for a Leu residue has been reported in two other FMTC families and associated with a low penetrance and a mild phenotype (Bolino et al, 1995). To determine whether this genotype-phenotype correlation applies also to the mutation reported here, experiments in an in vitro expression system are required (Santoro et al, 1995;Pasini et al, 1995).…”

A new syndrome: Heart defects, laryngeal anomalies, preaxial polydactyly, and colonic aganglionosis in sibs

“…Ten to forty percent of patients with familial Hirschsprung disease were found in one study to have an RET mutation. 13 In analysis of a large kindred, a G-to-T transversion mutation in exon 4 of the endothelin-B receptor is associated with Hirschsprung disease.I4 Twenty-one percent of the heterozygotes and 74% of homozygotes with the mutation in the endothelin-B receptor had aganglionosis of the intestine. A targeted interruption of the mouse endothelin-3, a ligand of the endothelin-B receptor, caused megacolon.15 This and other findings suggest that interaction of endothelin-3 and its receptors is crucial for neural crest-derived cell lineages.I6 Mice, in which the endothelin-B receptor gene had been deleted, showed a phenotype similar to that of our patients, i.e., intestinal aganglionosis, cardiac defects, and cleft liplcleft palate.…”